4&#39;-deoxyambutyrosin a and b

ABSTRACT

AN ANTIBIOTIC COMPLEX DESIGNATED BU-1975 IS FERMENTED FROM THREE SUBSPECIES OF THE MICROORGANISM NAMED BACILLUS CIRCULARS. THE ANTIOBIOTIC COMPLEX IS COMPRISED OF ARABUTYROSIN A. AMBUTYROSIN B, 4&#39;&#39;-DEOXYAMBUTYORISN A. 4&#39;&#39;-DEOXYBUTYROSIN B AND A PEPTIDE-LIKE FRACTION DESIGNATED B.

United States Patent O 3,826,802 -DEOXYAMBUTYROSIN A AND B Hiroshi Kawaguchi, Tokyo, Kei-Ichi Fujisawa, Kamifukuoka, Hiroshi Tsukiura, Tokyo, and Masataka Konishi, Yokohama, Japan, assignors to Bristol-Myers Company, New York, N X. No Drawing. Filed Sept. 13, 1972, Ser. No. 288,623 Int. Cl. A61k 21/00; C07c 129/18 U.S. Cl. 260-210 AB 12 Claims ABSTRACT OF THE DISCLOSURE An antibiotic complex designated Bu-1975 is fermented from three subspecies of the microorganism named Bacillus circulans. The antibiotic complex is comprised of ambutyrosin A, ambutyrosin B, 4'-deoxyambutyrosin A, 4-deoxybutyrosin B and a peptide-like fraction designated B.

BACKGROUND OF THE INVENTION 1. Field of the Invention The antibiotic fractions 4-deoxyambutyrosin A (C and 4'-deoxyambutyrosin B (C are new and novel compounds.

2. Description of the Prior Art The most pertinent prior art is U.S. Pat. No. 3,541,078 which discloses and claims the products generically known as ambutyrosin A and B. I

SUMMARY OF THE INVENTION Antibiotic complex Bu-1975, is fermented from Bacillus circulans subsp. n. croceus, Bacillus circulans subsp. n. proteophilus, or Bacillus circulans subsp. n. biotinicus. The two novel components of the complex claimed herein are 4deoxyambutyrosin A (C having the formula or: i c' c im g and 4'-deoxyambutyrosin B (C having the formula OH OH Component C is chemically known as N -(4-amino-2- hydroxybutyryl)-4-O-(2,6 diamino 2,4,6 trideoxy-D- glucopyranosyl)--O-D-xylofuranosyl 2 deoxystreptamine.

Component C is chemically known as N -(4-amino-2- hydroxybutyryl)-4-O-(2,6 diamino 2,4,6 trideoxy-D- ice glucopyranosyl)-S-O-D-ribofuranosyl 2 deoxystreptamine.

COMPLETE DISCLOSURE This invention relates to a new and novel antibiotic complex called Bu-1975 and to two novel components isolatable therefrom. The antibiotic complex, Bu-1975, has been isolated from the fermentation broth of three strains of Bacillus circulans, designated as Strain Nos. C308-B4, C436-B1 and C532-B2 in the Bristol-Banyu culture collection. The antibiotic is a complex of at least five bioactive components, A A B, C and C Components A and A were identified with ambutyrosins A and B [U.S'. Pat. No. 3,541,078 and Tetrahedron Letters, 28, pp. 2625- 2628 (1971)] respectively and component B was a solvent-extractable antibiotic with peptide-like properties.

Components C and C are new 'aminoglycoside antibiotics. C was found to be composed of D-xylose, Z-deoxystyreptamine, L-( -amino a hydroxybutyric acid and 2,6-diamino-2,4,6-trideoxy-D-glucose, a new deoxy amino sugar, and has been determined to be 4'-deoxyambutyrosin A which has the structure C was found to be composed of D-ribose, 2-deoxystreptamine, L-(-)-'y-amino -a hydroxybutyric acid and 2,6-diamino2,4,6-trideoxy-D-glucose and hence it is 4'-deoxyambutyr0sin B which has the structure OH OH Components C and C exhibit broader spectrum of antibacterial activity than kanamycin and ambutyrosin, both inhibiting some kanamycin and/or ambutyrosin-resistant organisms.

The complex is fermented from either of three subspecies of the microorganism Bacillus circulans.

A culture of each of the livng organisms has been deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 and have been assigned the following names and catalog numbers:

Bacillus circulans subsp. n. croceus (Strain No. C308- B4) A.T.C.C. 21820.

Bacillus circulans subsp. n. proteophilus (Strain No. C436-B1) A.T.C.C. 21821. I

Bacillus circulans subsp. n. biotinicus (Strain No. C532- B2 (A.T.C.C. 21822.

The morphological and cultural characteristics of these strains are shown in Table 1. The physiological reactions and carbon sources utilization of the strains are shown In view of the morphological, cultural and physiologiin Tables 2 and 3, respectively. cal characteristics described in these tables, all of the TABLE 1 [Morphological and cultural characteristics of Bu-1975 producing organisms] Vegetative cells Beds, 0.4 to 0.6 by 1.5 to 4.0 microns, Rods, 0.6 to 0.8 by 2.0 to 6.0 microns, Rods, 0.4 to 0.6 by 1.0 to 5.0 microns P with ends rounded, not in chains. with ends rounded, not in chains. with ends rounded, not in chains. Some palisade arrangement. Gram- Gram-variable, mostly negative. Gram-variable, mostly negative. variable, mostly negative.

Spores Oval to ellipsoidal, 0.9 to 1.4 by 1.4 to 2.2 Oval to ellipsoidal, 0.9 to 1.2 by 1.3 to 2.2 Oval to ellipsoidal, 1.0 to 1.4 by 1.5 to 2.4 microns; terminal to sub-terminal, microns; paracentral to terminal, microns;terminal, spore-wall thick and spore-wall thick and easily stained. spore-wall thick and easily stained. easily stained.

Sporangia Definitely swollen and racket-shaped..- Degriitey swollen and spindle to racket- Definitely swollen and racket-shaped.

s ape Motility Motile cell not found..... Mo Motile.

Nutrient agar slant".-- Growth abundant, thick, opaque, Growth late, moderate, thin, opaque, Growth abundant, thick, opaque,

smooth, viscous andcreamy becoming smooth, viscous and creamy. smooth, slightly viscous and whitish.

pale-orange with age. Glucose asparagiue agar Moderate growth. Raised, serru-trans- Scantgrowth,thin,translucent, smooth, No or scant growth.

slant. lucent, smooth, glistening, wrinkled, viscid and colorless.

viscid and slight-yellow.

Glu s broth pH 5.3-5.0 (37C.). Lightly viscous pH 'I.5-8.0 (25 0.). Viscous pellicle. pH 6.8-5.8 (37 0.). No pellicle. Light pellicle or ring growth. Produced Light turbidity and sediment. turbidityaud heavysedirnent. Glucose turbidity and sediment. broth plus biotine: pH 8.0-9.0. Complete pelhcle. Turbidity and light sediment.

Colony on nutrient agar. Circular, raised to domed with entire Circular or slightlyirregular, raised with Flat to alittle raised with slightly irregumargin. Opaque with smooth and irregular margin. Opaque with smooth lar margin. Opaque with smooth glistening suriace. Size: 1.0-3.5 mm. surface. Size: 1.0-4.0 mm. (in dia.). surface. Size 0.8-4.0 mm. (in dia.). (in dia.). Viscous and pale-orange. No Viscous and creamy. No satellite Slightly viscous and creamy white. N o

satellite colony. colony. satellite colony. Growth temperature:

Optimum 37 C 30 C 45 C. Moderate growth-... 28-45 C 2237 28-50 0. Restricted growth. 0., 48 0--. 20 0., 40 0- 0., 53 0. Scant growth 18 0., 50 C-.. 18 0., 42 0-.. 20 0., 55 0. No growth.-." 15 0., 53 C... 12 0., 45 C. 15 0., 58 0. Oxygen demand Aerobic Aerobic Aerobic. N aCl broth:

Growth 04% NaCl (Ii-3% NaCl 02% NaCl. No growth. 2% NaCl 4% NaCl I. 3% NaOl. Milk Viscous, thick pale orange pellicle with Viscous, thick pellicle with viscous sedi- Viscous, thick, faint-orange pellicle ith viscous sediment. No other change. ment. No other change. scant sediment. Coagulated without peptonization. pH slightly alkalized. Gelatin stab Viscid, pale-orange pellicle. Liquefied Visc1d,creamy pellicle. Liquefied gelatin Viscid, whitish pellicle. Liquefied gelatin gelatin completely after one week. completely after one week. completely after one week. Growth factor Nicotinic acid, biotine and complex of Nicotinic acid, biotine and complex of Biotine is essential for growth.

amino acids are accelerative for amino acids are accelerative for growth. growth.

TABLE 2 three strains were concluded to belong to species of Bacillus circulans. Significant difierences of each of the Ph l lr t' I We ogma m ions] three strains from the typical B. cu'culans are summarized Test C308-B4 C436-Bl 0532-132 below:

b h d t N t N t' N t' 833305300051? F553;? Pitta? P5505? Strain (3308-134- 1 d o io s). (st s)- ot c ng). Color of Colonyl: Pale orange. s t gn'ciiii dr i ii i '1 P iiiirzIlI PSEiiivZIII rgs iiix' Growth factor: Nicot n acid, biotin and amino (Iod e eac lo (O e Es g ga g ga acids complex accelerate the growth.

e O Citrate utilization N gativeuu N gativcnu Negative. suga'r utlllzatloni L'Arablllose and D-XY1OS8 are not Nitrate fronts nitrate. g ositiveflu Il Iosit1i; ve llggsitve ut l zed,

e 1V8- 0 3 lV9 8. l i it a l s e i ar i t ibn. Pos i tiveun- P0itive-- Pos itive. Stram (2436431: Oxidase reaction Negative..-. Negat v Negative' Glucose broth: Weakly alkaline reaction (pH 7.5-

8.0) after 3-5 days. TABLE 3 Growth temperature: Lower optimum temperature [Acid production from carbohydrates] G g w h at i) row ac or: icotinic aci iotin and amino 030834 643mm 653232 acids complex accelerate the growth. 1 mi 1 11 III 1 11 111 Strain CS32-B2: GlycemL i Growth factor: Biotin is essential for the growth. L-arabinese l- Sugar utilization: L-Arabmose and D-Xylose are not utilized. i 1+?- 1 l i l I rm b diif hfll b n view 0 e a ove erences t e o owin su 6O g I i I It I 1 1' I I species names were proposed for the three strains of i i i il: i+ i i f Bu-1975 producing organism. I iii ii'+ Bacillus circulans subsp. n. croceus (Strain C308-B4). i i Bacillus circulans subsp. n. proteophilus (Strain C436- I I I I I I I I I Bacillus circulans subs n. biotinicus Strain C532-B2 I I AfltlblOilC PIOdllCtlOIl A well-grown agar slant of the Bu-1975-producing orotis j' iei i iii o oii'ig i zii'- g 8 LE g sm Was u ed to inoculate the seed medium containg 4' 1,. 8.2- 2,005 a witilOmc.

eacli oi aspartate, glutamate and tryptophan 1 meg/ml. of nic tiiiic mg glucose 05% polypeptone 02% yeast extract 802% 0.0? e .lnihfiilsigtyinNH C1 7 KHPO 7 M SO H O 0.05 /a K HPO and 0.05% MgSO -7H O, the pH beasa eium 4 2 ,0.05 g 4-72,

0.057 NaCl, 0.1% 0000;, 0.01% yeast extract, 1. 5% Bacto-agar with 1 mg atdlustsd to [Before stenhzanon' The Seed culture mcg. mi. oi nicotinlc acid, 0.05 incgJml. oi biotin. was incubated at 37 C. for 24 hours on a rotary shaker Basal Medium n1: Pepmne bmth- (250 r.p.m.), and 2 ml. of the growth was transferred to 3,826,802 6 100 ml. of the fermentation medium in a 500-ml. Erlen- Physico-chemical Properties of Components C and C meyer flask, which has a composition of 3% soybean, meal 2% corn starch, 1% CaCO and 0.33% MgSO -7H O. Antibiotic production reached a maximum after 3-6 days shaking at 28 C.

The antibiotic activity in the fermentation broth was determined by the paper disc-agar difiusion assay using Bacillus subtilis PC1219 and Klebsiella pneumoniae A20680. All components of Bu-1975 complex (A A B, C and C showed activity against B. subtilis PC1219 Components C and C are white amorphous solid bases, which are readily soluble in water, slightly soluble in methanol and ethanol, and practically insoluble in nbutanol, acetone and other organic solvents. Both components give positive reactions with ninhydrin and anthrone reagents but are negative in Tollens, Fehling and Sakaguchi reactions.

An analytical sample of C was isolated in a form of 10 dicarbonate, which melted at about 155 C. (decomp.),

but only two components, C and C were active against 11:25: +2630 (c. 1'0 water) and analyzed as K. pneumoniae A20680.

The productivity of the C components relative to the 21 41 5 11' 2 a other components were different among the strains. In A l. Ca1cd.: C, 41.63; H, 6.83; N, 10.55. Found:

the shaking flask fermentation, strain C532-B2 produced C, 4152; H, 647; N,

50-100 meg/ml. of the C components which represented It gave tetra N acetate 5 C 24.s about 30-50% of the total bio-activity assayed by B. sub- (a 05, Water), which analyzed as tilis. Other strains were less productive of the C components than strain 0532-132. C21H41N5O11(C2H2O)4'3/2H2O Anal.Calcd.: C, 47.40; H, 7.13; N, 9.53. Found: C,

Isolation and Purification 47.30; H, 7A7; N 9.65.

The harvested broth was filtered using filter aid and the cozgnponent melted at 172478 (decomp), bio-activity in the filtrate (pH 8.0) was absorbed by a [MD =+0 water) and analyzed as column of Amberlite IRC-50 (NH form). The column F cmlslnlqsolrZHZCO3 was washed with water and then developed by 1 N NH OH solution. The active eluates were combined, confl Found! centrated in vacuo and extracted with n-butanol to re- C, 41.22; H, 6.67; N, 10.82. The tetra-N-acetate of C move component B in the concentrate. The aqueous layer was also prepared, -P- C, ]D

was separated and applied on a column of Amberlite water), and analyzed as C H N O '(C2 2O) -H 0. CG. 50 (NI-14+ form) The column was washed by water 30 Anal.Calcd.: C, 47.99; H, 7.08; N, 9.65. Found: C, and NM NH OH solution successively, and the activity 48.09; 7.2 N, 9.42.

was eluted by N/2 NH OH, the eluate being collected frac- AmiPiotics Q1 and 2 5h0w end absorption y in the tionally. The active components were eluted in the orultra V1018? Thfi Infrared p a of C and d of A A C2 and C1, though with considerable Over- C are quite similar to those of ambutyrosin A and B.

lap of the components, and the complete separation of The 9 magnetic spectrum (NMR of c1 shows two each component was achieved after repeating the CG-SO anomeflc P s a 5 5-28 (s) and 6.10 (d, J=3.5Hz column chromamgraphy p.p.m., the lower-field slgnal being different from that in As shown in Table 4 two TLC (thin layer chromatog the NMR spectrum of C which shows the anomeric proraphy) systems, 8-110 and S-117, were found suitable 2 at 5 (s) and 5- 3 ]=3'5HZ') to difierenfiate components A1 and A2 from components difference 1n the chemical shift of the second anomenc 1 and 2 and System S415 when developed for 16 protons has been seen in the NMR spectra of ambutyrosin A and B. The comparative NMR data of C and C along z enabled the separauon of A1 from and c1 from with ambutyrosin A and B are shown in Table 5. It is also ho th 1 Components A; and A; were identified with ambutyro- S wn m e Table that ram) of mtegra protons at the higher (6 1.2-2.4 ppm.) and the lower (6 2.5-4.4 8111s A and B respectively, by the physico-chemrcal proph 1 hi ion i 6; 18 for C and C fifties IR and and antibacterial pectrum. in contrast to the ratio of 4:19 for ambutyrosin A and B.

TABLE 5 Comparative NMR data of Components Cl and C: with Ambutyrosins A and B mHz, in D20 pH 2.0)

Number of protons and type of signals Chemical shift (6, ppm.) Bu-1975 C1 Bu-1975 C2 Amhutyrosin A Ambutyrosin B 6H (m) 4H (m) 4H (m) .16 18H (m) 19H (m) 19H (m) 1 z. 1 d, =3.6 Hz. 6.10 1H d, J=3.5 Hz.) 1H (d, J=3.6 Hz.)

The following descriptions are mostly confined to components C and C of antibiotic Bu-1975. Blologlcal Actwmes Antiba te e 'nim 1 The antibiotic complex which was identified with ambutyrt n31 trum The ml um mhlbliory osins A and B had been isolated in our screening program and concen ra of 1 and 2 were determmed was employed as reference in the present study.

TABLE 4 [TLC of Bu-1975 components] System Plate Solvent system A1 A2 C1 C2 Silica gel CHClr-MeOH28%NH OHH2O (1:4:2:1) 0.41."-.. 0.41"--- 0.51..." 0.51. do CHCl3-MeOH28%NH4OH (1:3:2) 0.20 0.20--- 0.26"--. 0.26. 8-115 Alumina CHCl MeOH-28%NH4OH (2:1:1) 3.7 cm-.- 0.2 cm-.- 5.3 cm... 0.5 cm.

1 Three times development. 2 16 hours development, location is shown in cm. from the origin.

In Vivo Activity and Toxicity Component C was evaluated in vivo comparatively with ambutyrosin A n experimental infections of mice. The pathogenic bacteria employed were S. aureus Smith, E. coli NIH] and pneumonz'ae A20680, the last one being the kanamyclnand ambutyrosin-resistant organism. M1ce were challenged intraperitoneally with the pathogens in a 5% suspension of hog gastric mucin, and the challenge dose was controlled to be 100 x LD A single subcutaneous treatment with the antibiotic was made immediately after the bacterial challenge. Group of five mice was used for one dosage level and the animals were observed for five days to determine the median curative dose (CD The results of the in vivo experiments are shown in Table 8. C afforded excellent protection in mice against all of the three infections tested. Ambutyrosin A showed comparable in vivo activity to that of C against the sensitive S. aureus and E. coli infections but was mactive against the resistant K. pneumoniae infectlon.

The acute toxicity of C was determined in mice. The intravenous LD was 720 mg./kg. for the sulfate of C and greater than 1000 mg./ kg. for the free base form. In a comparative experiment the intravenous LD of kanamycin sulfate and ambutyrosin sulfate were found to be 280 mg./kg. and 720 mg./kg., respectively.

TABLE 8 [In vivo activity of S. aureus Smith *No. survived/No. infected.

Norn.-Determined by the method of J. 1. Reed et al., American J. Hygiene, 27, 493-497 (1938).

In recent years there has been remarkable progress in elucidating the mechanisms of R-factor mediated resistance to the aminoglycoside antibiotics. These resistant organisms are now known to produce bacterial enzymes which inactivate aminoglycoside antibiotics by acetylation 5 6 phosphorylation 9 11 12 and adenyla- 5 Umezawa, H.; M. Okanishi, R. Utahara, K. Maeda and S. Kondo: Iso1ation and structure of kanamycin rnactivated by a cell free system of kanamycin resistant E. colt. J. Antiblotics, Ser. A 20 136-141. 1967.

Okanishi, M.; S. Kondo, R. Utahara and H. Umezawa Phosphorylation and inactivation of aminoglycosidic antibiotics by E. coli carrying R factor. J. Antlbiotics 21 (1): 13-21, 1968.

Benveniste, R.; and J. Davies: Enzymatic acetylation of aminoglycoside antibiotics by Escherichia 00 carrying an R factor. Biochemistry (10) 1787-1796, 1971.

tion 13 14 Kanamycin phosphorylase is known to react with kanamycins, nemoycins and paromomycins at the 3'-hydroxy group to yield bio-inactive, phosphorylated products Gentamicins C C and C tobramycin and dideoxykanamycin B which have no hydroxy group in the 3'-position, are incapable of the phosphorylative inactivation. Ambutyrosin is not inactivated by the kanamycin phosphorylase (unpublished data) though it has a 3-hydroxy group, and the resistance of ambutyrosin to this enzymatic inactivation is supposed to be due to the acyl substitution of l-amino function with L-(-)-'yamino-a-hydroxybutyric acid. This is also the case with C and C which showed activity against the kanamycin-rcsistant organisms that produce kanamycin phosphorylase. This is quite natural since C and C have the structure of 4'-deoxyambutyrosin A and B.

A further interesting finding was that C and C inhibited growth of K. pneumoniae A20680 and E. coli A20683 which were known to inactivate gentamicin C componcuts and 3',4'-dideoxykanamycin B 15 by adenylation, and kanamycin, neomycin and streptomycin by phosphorylation As can be seen in Tables 6 and 7, ambutyrosin lacks activity in this type of resistant organisms.

Antibiotic complex Bil-1975, and particularly the purified components C and C are valuable as antibacterial agents, nutritional supplements in animal feeds, therapeutic agents in poultry and animals, including man, and are especially valuable in the treatment of infectious diseases caused by Gram-positive and Gram-negative bacteria.

C and C when administered orally are useful as an adjunctive treatment for preoperative sterilization of the bowel. Both aerobic and anaerobic flora which are susceptible to these drugs are reduced in the large intestine. When accompanied by adequate mechanical cleansing, they are useful in preparing for colonic surgery.

C and C are effective in the treatment of systemic bacterial infections when administered parenterally in the dosage range of about 250 mg. to about 3000 mg. per day in divided doses three or four times a day. Generally the compounds are effective when administered at a dosage of about 5.0 to 7.5 mg./kg. of body weight every 12 hours.

Brzezinska, M.; R. Benveniste, J. Davies, P. J. Daniels and J. Weinstein Gentamicin resistance in strains of Pseudomonas aerugihosa mediated by enzymatic N-acetylation of the deoxystreptamine moiety. Biochemistry, 11 761-765, 1972.

9 Umezawa, H.; M. Okanishi, S. Kondo, K. Hamana, Utahara, K. Maeda and S. Mitsuhasi: Phosphorylative inactivation of aminoglycosidic antibiotics by Escherichia coli carrying R factor. Science 157 1559-1561, 1967.

Okanishi, M.; S. Kondo, R. Utahara and H. Umezawa: Phosphorylation and inactivation of aminoglycosidic antiggostgcs by E. coli carrying R factor J. Antibiotics 21: 13-21,

11 Umezawa, H. 0. D01, M. Ogura, S. Kondo and N. Tanaka. Phosphorylation and inactivation of kanamycin by Pseudomomzs aerugihosa. J. Antibiotics 21 154-155, 1968.

Ozanne, B.; R. Benveniste, D. Tipper and J. Davies: Aminoglycoside antibiotics: Inactivation by phosphorylation in Escherichia 00 carrying R factors. J. Bacteriol. (2) 1144-1146, 1969.

Umezawa, H. S. Takasawa, M. Okanishi and R. Utahara Adenylylstreptomycin, a product of streptomycin inactivated by E. 0011 carrying R factor. J. Antibiotics 21: 81-82, 1968.

Benveniste, R. and J. Davies: R factor mediated gentumicin resistance: A new enzyme which modifies aminoglycoside antibiotics. FEBS Letters 14 (5): 293-296, 1971.

Yagisawa, M.; H. Naganawa, S. Kondo, M. Hamada, T. Takeuchi and H. Umezawa: Adenyldideoxykanamyci-n B, a product pt the inactivation of dideoxykanamycin B by Eschericgml EOZL carrying R factor. J. Antibiotics. 24 (12) 911-912,

16 Davies, J.; M. Brzezinska and R. Benveniste: R factors: Biochemical mechanisms of resistance of aminoglycoside antibiotics. Ann. N.Y. Acad. Sci. 182: 226-233, 1971.

Koch, K. F. and J. A. Rhoades: Structure of nebramycin factor 6, a new aminoglycosidic antibiotic. Antimicrobial Agent Chemotherapy 1970 309-313, 1971.

Umezawa. H. S. Umezawa. T. Tsuchiya. and Y. Okazaki: 3, 4-Dideoxykanamyc n B active against kanamycin-resistant Eschemchm coli and Pseudommtas aerugt'nosa. J. Antibiot. 21;: 485-487. 1971.

19 Martin, C. M. N. S. Ikari, J. Zimmerman and J. A. Waitz A virulent nosocomial Klebsiella with a transferable R factor of Genarnrcin: Emergence and suppression. J. Infect. Dis. 124 (Supplement) $24-$29. 1971.

K pneumomae A20680 is originated from the strain of Klebszellc type 22 No. 3038 (see footnotes 14 and 19).

E. 0011. A20683 is originated from the strain of E. 0011' JR66/W677 (see footnote 14).

A preferred embodiment of the present invention is a compound selected from the group comprising OH OH or a pharmaceutically acceptable acid addition salt thereof.

A more preferred embodiment is the compound designated herein as the antibiotic C or a pharmaceutically acceptable acid addition salt thereof.

Another preferred embodiment is the compound designated herein as the antibiotic C or a pharmaceutically acceptable acid addition salt thereof.

Most preferred embodiments are the monosulfate, disulfate, unhydrated, monohydrated and polyhydrated forms of the antibiotics C and C For the purpose of this disclosure, the term nontoxic pharmaceutically acceptable acid addition salt shall mean a mono, di-, trior tetrasalt formed by the interaction of one molecule of C or C with 14 moles of a nontoxic, pharmaceutically acceptable acid. Included among these acids are acetic, hydrochloric, sulfuric, maleic, phosphoric, nitric, hydrobromic, ascorbic, malic and citric acid, and those other acids commonly used to make salts of amine containing pharmaceuticals.

Another preferred embodiment is the process for the preparation of the antibiotics having the formula which comprises fermenting the antibiotics using the cultures designated Bacillus circulans subsp. n. croceus (A.T. C.C. 21820), Bacillus circulans subsp. n. proteophilzrs (A.T.C.C. 21821), or Bacillus circulans subsp. 11. Biotinicus (A.T.C.C. 21822) by aerobic fermentation and subsequently isolating the antibiotic components.

Amberlite IRC 50 is the trade name for a weakly acidic cationic exchange resin of a carboxylic-polymethacrylic type.

Amberlite CG 50 is the trade name for the chromatographic grade of a weakly acidic cationic exchange resin of a carboxylic-polymethacrylic type.

Example 1 Agar slant culture of B. circulans strain C532-B2 Was used to inoculate ml. of medium No. YGP-1 (1.5% glucose, 0.5% polypeptone, 0.2% yeast extract, 0.05% K HPO and 0.05% MgSO -7H O) in a 500 ml. Erlenmeyer flask. The seed culture was incubated at 37 C. for 24 hours on rotary shaker (250 rpm.) and each 2 ml. of the growth was transferred to 100 ml. of fermentation medium No. 132 (3% soybean meal, 2% corn starch, 1% CaCO and 0.33% MgSO -7H O). After six days shaking culture at 28 C., the paper disc (8 mm.) assay of the fermentation broth showed 25 mm. inhibition zone on B. subtilis PC1219 plate and 16 mm. zone of K. pneumoniae A20680 plate. Combined fermentation broth (200 flasks, 171) was filtered and absorbed by a column of Amberlite IRC-50 (NH form, 500 ml.). The column was Washed with Water and then eluted by 2.2 l of 1N NH OH solution. The active eluates were combined, concentrated in vacuo and extracted with n-butanol. Evaporation of the butanol extract gave 1.3 g. of crude solid (component B). The aqueous layer was concentrated in vacuo to about 30 ml. which was applied on a column of Amberlite CG-SO (NH form). The column was washed with 500 ml. of N/10 NH OH and 700 ml. of NM NH OH successively, and the bioactivity was eluated by N/2 NH OH solution. The eluate was collected fractionally and traced by bio-assay, ninhydrin reaction and TLC. The active components were eluted in the order of A A C2 and C Esti- Volurne, mated Solid Tube Nos: ml. TLC ratio mgr 69-87 200 A2+A1 4:6 228 88-111 250 A +C 3:7 193 112-159 500 C 201 Repeated column chromatography (CG-50, NH form) of the second and third solids gave pure preparations of mg. of C and 100 mg. of C respectively.

Example 2 Example 3 B. circulans strain C308-B4 was used as a seed culture and shake flask fermentation was conducted as in Example 1. The harvested broth (18 1.) contained 75 mg. of component A (A +A 6.4 g. of component B and 75 mg. of component C (C l-C Example 4 B. circulans strain C436-B1 was used as a seed culture and shake flask fermentation was conducted as in Example 1. The harvested broth (20 1.) contained 71 mg. of

13 component A (A +A 4.1 g. of component B and 66 mg. of component C (C +C Example Submerged and aerated fermentation experiment was carried out in 20-1. jar fermentors. B. circulans strain C532-B2-H48 was used to inoculate l. of medium No. YGP-l (pH 7.2 after sterilization). The seed culture was stirred at 250 r.p.m. at 35 C. with aeration rate of 10 l./min., and gave vigorous growth after 11 hours (pH 6.0). At 11.5 hours, 1 l. of the seed culture was transferred to 10 1. of sterilized production medium No. 132. The fermentation was carried out at 28 C. with aeration rate of 11.5 l./min. Frequent additions of silicone antifoam (KM-70) were necessary to control the excessive foaming. A peak potency of the broth was attained at 70 hours (pH 8.2), and the differential assay using B. subtilz's PCI 219 and K. pneumoniae A20680 showed 79 meg/ml. of component A (A +A and 49 meg/ml. of component C (C +C Example 6 Larger scale fermentation was carried out in pilot plant tanks capable of fermenting 100 l. and 300 1. volumes. The fermentation conditions were similar to those of Example 5 except that the agitation rate was 180 r.p.m. and the incubation temperature of production medium was 32 C. After 50 hours fermentation (pH 8.7), the broth potency reached 95 meg/ml. of component A and 45 mcg./ ml. of component C.

Example 7 Harvested broth from two 300-1. tanks (650 1.) containing about 20 meg/ml. of component C was filtered at pH 8.5 and stirred with 8.6 l. of Amberlite IRC-5O (NHJ form). The resin was separated, washed with 80 l. of water and then eluted batchwise with 1 N NH OH solution (10 l. x 3). The eluates were combined and concentrated in vacuo at 3540 C. to about 500 ml. volume which contained 11.3 gram unit of component A and 7.3 gram unit of component C. The concentrate was extracted with n-butanol to remove contaminated component B, and the aqueous layer was stirred with 200 ml. of Amberlite CG50 (NH form). The resin was separated, washed with 5 l. of water and then placed on a top of CG50 column (NH form, 1500 ml.). The column was developed with 7 l. of N/4 NH OH. The activity was eluted with N/ 2 NH OH and the eluates were collected fractionally, yielding 7.2 g. of component A from tube Nos. 491-800 and 4.2 g. of component C from tube Nos. 931-1370. Mixture of components A and C, 1.8 g., was recovered from tube Nos. 801-930.

We claim:

1. A compound selected from the group comprising or a pharmaceutically acceptable acid addition salt thereof.

2. The compound of claim 1 having the formula or a pharmaceutically acceptable acid addition salt thereof.

3. The compound of claim 1 having the formula or a pharmaceutically acceptable acid addition salt thereof.

4. The monosulfate salt of the compound of claim 2.

5. The monosulfate salt of the compound of claim 3.

6. The disulfate salt of the compound of claim 2.

7. The disulfate salt of the compound of claim 3.

8. The mono or polyhydrates of the compounds of claim 1.

9. The mono or polyhydrates of the compound of claim 4.

10. The mono or polyhydrates of the compound of claim 5.

11. The mono or polyhydrate of the compound of claim 6.

12. The mono or polyhydrate of the compound of claim 7.

References Cited UNITED STATES PATENTS 3,541,078 11/1970 Woo et a1. 260-210 AB 3,661,892 5/1972 Shomura et al. 260-210 AB JOHNNIE R. BROWN, Primary Examiner U.S. Cl. X.R. -96; 424-181 

